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Protein synthesis is a cellular process essential for life. Ribosomes are responsible for protein synthesis in all cells. Ribosomes are composed of three RNA molecules (23S, 16S, and 5S ribosomal RNAs) and about 50 proteins. The ribosomal RNAs play a central role in protein synthesis by forming the active site for peptide bond formation and by interacting with messenger RNA, transfer RNA, and translational factors. Here we describe the functional role of the ribosomal RNAs in protein synthesis.

Ribosomes are the universally conserved ribonucleoprotein complexes that synthesize proteins. The two subunits of the eukaryotic ribosome are produced through a quasi-independent assembly-line-like pathway involving the hierarchical actions of numerous trans-acting biogenesis factors and the incorporation of ribosomal proteins. The factors work together to shape the nascent subunits through a series of intermediate states into their functional architectures. The earliest intermediate of the small subunit (SSU or 40S) is the SSU Processome which is subsequently transformed into the pre-40S intermediate. This transformation is, in part, facilitated by the binding of the methyltransferase Bud23. How Bud23 is released from the resultant pre-40S is not known. The ribosomal proteins Rps0, Rps2, and Rps21, termed the Rps0-cluster proteins, and several biogenesis factors are known to bind the pre-40S around the time that Bud23 is released, suggesting that one or more of these factors induce Bud23 release. Here, we systematically examined the requirement of these factors for the release of Bud23 from pre-40S particles. We found that the Rps0-cluster proteins are needed but not sufficient for Bud23 release. The atypical kinase/ATPase Rio2 shares a binding site with Bud23 and is thought to be recruited to pre-40S after the Rps0-cluster proteins. Depletion of Rio2 prevented the release of Bud23 from the pre-40S. More importantly, the addition of recombinant Rio2 to pre-40S particles affinity-purified from Rio2-depleted cells was sufficient for Bud23 release in vitro. The ability of Rio2 to displace Bud23 was independent of nucleotide hydrolysis. We propose a novel role for Rio2 in which its binding to the pre-40S actively displaces Bud23 from the pre-40S, and we suggest a model in which the binding of the Rps0-cluster proteins and Rio2 promote the release of Bud23.

Translation of the genetic code into proteins is realized through repetitions of synchronous translocation of messenger RNA (mRNA) and transfer RNAs (tRNA) through the ribosome. In eukaryotes translocation is ensured by elongation factor 2 (eEF2), which catalyses the process and actively contributes to its accuracy1. Although numerous studies point to critical roles for both the conserved eukaryotic posttranslational modification diphthamide in eEF2 and tRNA modifications in supporting the accuracy of translocation, detailed molecular mechanisms describing their specific functions are poorly understood. Here we report a high-resolution X-ray structure of the eukaryotic 80S ribosome in a translocation-intermediate state containing mRNA, naturally modified eEF2 and tRNAs. The crystal structure reveals a network of stabilization of codon–anticodon interactions involving diphthamide1 and the hypermodified nucleoside wybutosine at position 37 of phenylalanine tRNA, which is also known to enhance translation accuracy2. The model demonstrates how the decoding centre releases a codon–anticodon duplex, allowing its movement on the ribosome, and emphasizes the function of eEF2 as a ‘pawl’ defining the directionality of translocation3. This model suggests how eukaryote-specific elements of the 80S ribosome, eEF2 and tRNAs undergo large-scale molecular reorganizations to ensure maintenance of the mRNA reading frame during the complex process of translocation., Structural analysis of the Saccharomyces cerevisiae 80S ribosome trapped in an intermediate translocation state shows stabilization of codon–anticodon interactions by eukaryote-specific elements of the 80S ribosome, eEF2 and tRNA and demonstrates a major role for eEF2 in maintaining the directionality of translocation.

Nsp1 of SARS-CoV-2 regulates the translation of host and viral mRNAs in cells. Nsp1 inhibits host translation initiation by occluding the entry channel of the 40S ribosome subunit. The structural study of the Nsp1-ribosomal complexes reported post-termination 80S complex containing Nsp1, eRF1 and ABCE1. Considering the presence of Nsp1 in the post-termination 80S ribosomal complex, we hypothesized that Nsp1 may be involved in translation termination. Using a cell-free translation system and reconstituted in vitro translation system, we show that Nsp1 stimulates peptide release and formation of termination complexes. Detailed analysis of Nsp1 activity during translation termination stages reveals that Nsp1 facilitates stop codon recognition. We demonstrate that Nsp1 stimulation targets eRF1 and does not affect eRF3. Moreover, Nsp1 increases amount of the termination complexes at all three stop codons. The activity of Nsp1 in translation termination is provided by its N-terminal domain and the minimal required part of eRF1 is NM domain. We assume that the biological meaning of Nsp1 activity in translation termination is binding with the 80S ribosomes translating host mRNAs and remove them from the pool of the active ribosomes.

Background: The majority of scientists, physicians, and healthcare professionals were trained with the paradigm: “antibiotics are for bacteria only !”, because they misunderstood the definition of the ribosome targeting antibiotics. In the context of the current worldwide COVID-19 pandemic, it might be useful to recall as precisely as possible the definition of the word antibiotic and provide evidence that some classes of antibiotics could offer excellent means to counteract viral infections via specific mechanisms. Methods: Molecular modeling and docking studies were used, as well as the tRNAox labeling reaction of the ribosomal protein eL42 in situ on human 80S ribosomes to demonstrate that cycloheximide and its thiosemicarbazone analogues bind to the catalytic Lys-53 residue of the human large subunit ribosomal protein eL42. Results: Comparison of the binding sites for Cycloheximide (CHX) and Sparsomycin (SPS) on the evolutionarily conserved E. coli bL12 and S. cerevisiae eL42 by means of molecular modeling and docking studies showed that: (i) SPS binds in proximity to the catalytic Lys-65 residue of the GANK motif of rp bL12 and to the catalytic Lys-55 residue of the GGQTKP motif of rp eL42; (ii) CHX failed to bind to the GANK motif, while the glutarimide moiety of SPS and CHX was found to make contact with Lys-55 of the GGQTKP motif of rp eL42. Conclusion: In this report, we demonstrate that cycloheximide and its thiosemicarbazone analogues are capable of inhibiting the human 80S ribosomes selectively through their binding to the ε-amino group of the side chain of Lys-53. As a consequence, these small-molecule inhibitors of translation are susceptible to exhibit antiviral activities by preventing the human ribosomes of the SARS-CoV-2 infected cells from synthesizing the viral proteins and enzymes.

Ribosomes are the macromolecular machines at the heart of protein synthesis; however, their function can be modulated by a variety of additional protein factors that directly interact with them. Here, we report the cryo-EM structure of human Ebp1 (p48 isoform) bound to the human 80S ribosome at 3.3 Å resolution. Ebp1 binds in the vicinity of the peptide exit tunnel on the 80S ribosome, and this binding is enhanced upon puromycin-mediated translational inhibition. The association of Ebp1 with the 80S ribosome centers around its interaction with ribosomal proteins eL19 and uL23 and the 28S rRNA. Further analysis of the Ebp1-ribosome complex suggests that Ebp1 can rotate around its insert domain, which may enable it to assume a wide range of conformations while maintaining its interaction with the ribosome. Structurally, Ebp1 shares homology with the methionine aminopeptidase 2 family of enzymes; therefore, this inherent flexibility may also be conserved.

The alkaloid agelastatin A (AGA) has raised great interest for its potent antitumor activity and intriguing molecular structure. This led to multifarious total syntheses and the antitumor activity was related to eukaryotic translation inhibition, unraveling the structure of the complex with the 80S yeast ribosome at atomic level. What remained unanswered is the relative weight of the various interactions, a hierarchy of great interest in planning improved antitumor agents in the AGA family. This has been investigated here along QM-MM in explicit water. What emerged was a high and constant HOMO-LUMO gap, speaking for a robust AGA-ribosome complex also in solution, within which AGA’s relaxed twisting ability showed up markedly. It became also clear that AGA’s ring A, despite its formal representation, has no pyrrole properties. Br-π and π-π stacking interactions and H-bonds with surrounding nucleotides, as from X-ray diffraction, are fully supported by MEP maps. But the main finding was that MD-driven dissection of the various contributions revealed a dominance of the entropically favored chelate H-bonding between AGA’s central amide system and U2869. In contrast, the X-ray diffraction assignment of C5-OH H-bonding to U2873 is not supported. Along similar lines it was found that interactions of the ribosome with the structural analog CEAA, where chlorine and ethyl replace AGA’s bromine and methyl, differ markedly, except as to the chelate bonding with U2869, which persists as a character of the class. This should be useful in the invention of improved translation inhibitors in the agelastatin class.

Graphical abstract, Highlights • DEER reveals the conformational variability of mRNA at the certain translation steps. • Elongation and termination complexes exist in 2 conformations in dynamic equilibrium. • The conformations of mRNA in 40S channel undergo no major change during termination., The conformation of mRNA in the region of the human 80S ribosome decoding site was monitored using 11-mer mRNA analogues that bore nitroxide spin labels attached to the terminal nucleotide bases. Intramolecular spin–spin distances were measured by DEER/PELDOR spectroscopy in model complexes mimicking different states of the 80S ribosome during elongation and termination of translation. The measurements revealed that in all studied complexes, mRNA exists in two alternative conformations, whose ratios are different in post-translocation, pre-translocation and termination complexes. We found that the presence of a tRNA molecule at the ribosomal A site decreases the relative share of the more extended mRNA conformation, whereas the binding of eRF1 (alone or in a complex with eRF3) results in the opposite effect. In the termination complexes, the ratios of mRNA conformations are practically the same, indicating that a part of mRNA bound in the ribosome channel does not undergo significant structural alterations in the course of completion of the translation. Our results contribute to the understanding of mRNA molecular dynamics in the mammalian ribosome channel during translation.

Arabidopsis REIL proteins are cytosolic ribosomal 60S-biogenesis factors. After shift to 10°C,reilmutants deplete and slowly replenish non-translating eukaryotic ribosome complexes of root tissue, while tightly controlling the balance of non-translating 40S- and 60S-subunits.Reilmutations compensate by hyper-accumulation of non-translating subunits at steady-state temperature; after cold-shift, a KCl-sensitive 80S sub-fraction remains depleted. We infer that Arabidopsis buffers fluctuating translation by pre-existing non-translating ribosomes beforede novosynthesis meets temperature-induced demands.Reil1 reil2double mutants accumulate 43S-preinitiation and pre-60S-maturation complexes and have altered paralog composition of ribosomal proteins in non-translating complexes. With few exceptions, e.g. RPL3B and RPL24C, these changes are not under transcriptional control. Our study suggests requirement ofde novosynthesis of eukaryotic ribosomes for long-term cold acclimation, feedback control ofNUC2andeIF3C2transcription and links new proteins, AT1G03250, AT5G60530, to plant ribosome biogenesis. We propose that Arabidopsis requires biosynthesis of specialized ribosomes for cold acclimation.Highlight of this studyREIL proteins affect paralog composition of eukaryotic ribosomes and suppress accumulation of 43S-preinitiation and pre-60S-maturation complexes, suggesting functions of ribosome heterogeneity and biogenesis in plant cold acclimation.

Eukaryotic ribosome and cap-dependent translation are attractive targets in the antitumor, antiviral, anti-inflammatory, and antiparasitic therapies. Currently, a broad array of small-molecule drugs is known that specifically inhibit protein synthesis in eukaryotic cells. Many of them are well-studied ribosome-targeting antibiotics that block translocation, the peptidyl transferase center or the polypeptide exit tunnel, modulate the binding of translation machinery components to the ribosome, and induce miscoding, premature termination or stop codon readthrough. Such inhibitors are widely used as anticancer, anthelmintic and antifungal agents in medicine, as well as fungicides in agriculture. Chemicals that affect the accuracy of stop codon recognition are promising drugs for the nonsense suppression therapy of hereditary diseases and restoration of tumor suppressor function in cancer cells. Other compounds inhibit aminoacyl-tRNA synthetases, translation factors, and components of translation-associated signaling pathways, including mTOR kinase. Some of them have antidepressant, immunosuppressive and geroprotective properties. Translation inhibitors are also used in research for gene expression analysis by ribosome profiling, as well as in cell culture techniques. In this article, we review well-studied and less known inhibitors of eukaryotic protein synthesis (with the exception of mitochondrial and plastid translation) classified by their targets and briefly describe the action mechanisms of these compounds. We also present a continuously updated database (http://eupsic.belozersky.msu.ru/) that currently contains information on 370 inhibitors of eukaryotic protein synthesis.

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure syndrome usually caused by heterozygous variants in ribosomal proteins (RP) and which leads to severe anemia. Genetic studies in DBA rely primarily on multigene panels that often result in variants of unknown significance. Our objective was to optimize polysome profiling to functionally validate new large subunit RP variants. We determined the optimal experimental conditions for B-cell polysome profiles then performed this analysis on 2 children with DBA and novel missense RPL5 (uL18) and RPL26 (uL24) variants of unknown significance. Both patients had reduced 60S and 80S fractions when compared with an unaffected parent consistent with a large ribosomal subunit defect. Polysome profiling using primary B-cells is an adjunctive tool that can assist in validation of large subunit RP variants of uncertain significance. Further studies are necessary to validate this method in patients with known DBA mutations, small RP subunit variants, and silent carriers.

Nanopores hold great potential for the analysis of complex biological molecules at the single entity level. One particularly interesting macromolecular machine is the ribosome, responsible for translating mRNA into proteins. In this study, we use a solid-state nanopore to fingerprint 80S ribosomes and polysomes from a human neuronal cell line and, Drosophila melanogaster cultured cells and ovaries. Specifically, we show that the peak amplitude and dwell time characteristics of 80S ribosomes are distinct from polysomes and can be used to discriminate ribosomes from polysomes in mixed samples. Moreover, we are able to distinguish large polysomes, containing more than 7 ribosomes, from those containing 2-3 ribosomes, and demonstrate a correlation between polysome size and peak amplitude. This study highlights the application of solid-state nanopores as a rapid analytical tool for the detection and characterization of ribosomal complexes.

Cytosolic 80S ribosomes contain proteins of the mature cytosolic ribosome (r-proteins) as well as proteins with roles in ribosome biogenesis, protein folding or modification. Here, we refined the core r-protein composition in Arabidopsis thaliana by determining the abundance of different proteins during enrichment of ribosomes from cell cultures using peptide mass spectrometry. The turnover rates of 26 40S subunit r-proteins and 29 60S subunit r-proteins were also determined, showing that half of the ribosome population is replaced every 3–4 days. Three enriched proteins showed significantly shorter half-lives; a protein annotated as a ribosomal protein uL10 (RPP0D, At1g25260) with a half-life of 0.5 days and RACK1b and c with half-lives of 1–1.4 days. The At1g25260 protein is a homologue of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role in the 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins; 26 were enriched more than 10-fold during ribosome enrichment. Some of these enriched proteins have known roles in translation, while others are newly proposed ribosome-associated factors in plants. This analysis provides an improved understanding of A. thaliana ribosome protein content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and how protein turnover can help identify r-proteins involved in ribosome biogenesis or regulation in plants.

The human tau is a microtubule-associated intrinsically unstructured protein that forms intraneuronal cytotoxic deposits in neurodegenerative diseases, like tauopathies. Recent studies indicate that in Alzheimer’s disease, ribosomal dysfunction might be a crucial event in the disease pathology. Our earlier studies had demonstrated that amorphous protein aggregation in the presence of ribosome can lead to sequestration of the ribosomal components. The present study aims at determining the effect of incubation of the full-length tau protein (Ht40) and its microtubule binding 4-repeat domain (K18) on the eukaryotic ribosome. Our in vitro studies show that incubation of Ht40 and the K18 tau variants with isolated non-translating yeast ribosome can induce a loss of ribosome physical integrity resulting in formation of tau-rRNA-ribosomal protein aggregates. Incubation with the tau protein variants also led to a disappearance of the peak indicating the ribosome profile of the HeLa cell lysate and suppression of translation in the human in vitro translation system. The incubation of tau protein with the ribosomal RNA leads to the formation of tau-rRNA aggregates. The effect of K18 on the yeast ribosome can be mitigated in the presence of cellular polyanions like heparin and tRNA, thereby indicating the electrostatic nature of the aggregation process.

We have recently developed an in vitro yeast reconstituted translation system, which is capable of synthesizing long polypeptides. Utilizing the system, we examined the role of eIF5A and its hypusine modification in translating polyproline sequence within long open reading frames. We found that polyproline motif inserted at the internal position of the protein arrests translation exclusively at low Mg2+ concentrations, and peptidylpolyproline-tRNA intrinsically destabilizes 80S ribosomes. We demonstrate that unmodified eIF5A essentially resolves such ribosome stalling; however, the hypusine modification drastically stimulates ability of eIF5A to rescue polyproline-mediated ribosome stalling and is particularly important for the efficient translation of the N-terminal or long internal polyproline motifs.

Human Ebp1 is a member of the proliferation-associated 2G4 (PA2G4) family and plays an important role in cancer regulation. Ebp1 shares the methionine aminopeptidase (MetAP) fold and binds to mature 80S ribosomes for translational control. Here, we present a cryo-EM single particle analysis reconstruction of Ebp1 bound to non-translating human 80S ribosomes at a resolution range from 3.3 to ~8 Å. Ebp1 blocks the tunnel exit with major interactions to the general uL23/uL29 docking site for nascent chain-associated factors complemented by eukaryote-specific eL19 and rRNA helix H59. H59 is defined as dynamic adaptor undergoing significant remodeling upon Ebp1 binding. Ebp1 recruits rRNA expansion segment ES27L to the tunnel exit via specific interactions with rRNA consensus sequences. The Ebp1-ribosome complex serves as a template for MetAP binding and provides insights into the structural principles for spatial coordination of co-translational events and molecular triage at the ribosomal tunnel exit., The ErbB3 receptor binding protein Ebp1 binds to ribosomes and is linked to translational control. Here, the authors present the cryo-EM structure of human Ebp1 bound to a non-translating 80S ribosome and find that Ebp1 blocks the tunnel exit and recruits the rRNA expansion segment ES27L to the tunnel exit.

We have created a bacterial semi-synthetic organism (SSO) that retains an unnatural base pair (UBP) in its DNA, transcribes it into mRNA and tRNA with cognate unnatural codons and anticodons, and after the tRNA is charged with a non-canonical amino acid, synthesizes proteins containing the non-canonical amino acid. Here, we report the first progress toward the creation of eukaryotic SSOs. After demonstrating proof-of-concept with human HEK293 cells, we show that a variety of different unnatural codon-anticodon pairs can efficiently mediate the synthesis of unnatural proteins in CHO cells. Interestingly, we find that there are both similarities and significant differences between how the prokaryotic and eukaryotic ribosomes recognize the UBP, with the eukaryotic ribosome appearing more tolerant. The results represent the first progress toward eukaryotic SSOs and in fact, suggest that such SSOs might be able to retain more unnatural information than their bacterial counterparts.

A number of regulatory nascent peptides have been shown to regulate gene expression by causing programmed ribosome stalling during translation. Nascent peptide emerges from the ribosome through the exit tunnel, and one-third of the way along which β-loop structures of ribosomal proteins uL4 and uL22 protrude into the tunnel to form the constriction region. Structural studies have shown interactions between nascent peptides and the exit tunnel components including the constriction region. In eukaryotes, however, there is a lack of genetic studies for the involvement of the constriction region in ribosome stalling. Here, we established transgenic Arabidopsis lines that carry mutations in the β-loop structure of uL4. Translation analyses using a cell-free translation system derived from the transgenic Arabidopsis carrying the mutant ribosome showed that the uL4 mutations reduced the ribosome stalling of four eukaryotic stalling systems, including those for which stalled structures have been solved. Our data, which showed differential effects of the uL4 mutations depending on the stalling systems, explained the spatial allocations of the nascent peptides at the constriction that were deduced by structural studies. Conversely, our data may predict allocation of the nascent peptide at the constriction of stalling systems for which structural studies are not done.

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splitting in vitro. Collectively, the data suggest that eEF2K defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons., Processing bodies are phase separated compartments enriched in translationally repressed mRNAs. Here, Smith et al. show that, in sensory neurons, eukaryotic elongation factor 2 kinase (eEF2K) plays key roles in the regulation of processing body abundance and the formation of translationally inactive ribosomes.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

Summary Conserved developmentally regulated guanosine triphosphate (GTP)-binding proteins (Drgs) and their binding partner Drg family regulatory proteins (Dfrps) are important for embryonic development, cellular growth control, differentiation, and proliferation. Here, we report that the yeast Drg1/Dfrp1 ortholog Rbg1/Tma46 facilitates translational initiation, elongation, and termination by suppressing prolonged ribosome pausing. Consistent with the genome-wide observations, deletion of Rbg1 exacerbates the growth defect resulting from translation stalling, and Rbg1 stabilizes mRNAs against no-go decay. Furthermore, we provide a cryoelectron microscopy (cryo-EM) structure of the 80S ribosome bound with Rbg1/Tma46 that reveals the molecular interactions responsible for Rbg1/Tma46 function. The Rbg1 subunit binds to the GTPase association center of the ribosome and the A-tRNA, and the N-terminal zinc finger domain of the Tma46 subunit binds to the 40S, establishing an interaction critical for the ribosomal association. Our results answer the fundamental question of how a paused ribosome resumes translation and show that Drg1/Dfrp1 play a critical role in ensuring orderly translation.

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit., The Henry/Henras group is supported by grants from ANR (ANR-20-CE12-0026) and funding from CNRS and University of Toulouse. R.A.M. is supported by grants from the Rectorat of Lebanese University. M.J. is supported by a Ph.D. fellowship from the Lebanese University and CIOES Organization. The group of J.d.l.C. is supported by the Spanish Ministry of Science and Innovation [PID2019-103859-GB-I00 AEI/ 10.13039/501100011033], and the Andalusian Regional Government (JA; BIO-271). J.C. was supported by a Ph.D. fellowship (PIF) from the University of Seville, and S.M.-V. is an academic research staff of the JA (PAIDI2020). M.T.B. and K.E.B. are supported by funding from the Deutsche Forschungsgemeinschaft (SFB860) and the University Medical Centre Göttingen.

PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. piRNAs originate from transposable element (TE) RNAs, long non-coding RNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, with the last being the least characterized of the three piRNA classes. Here, we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens. At the pachytene stage, when other co-translational RNA surveillance pathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes., Ribosome can mediate piRNA biogenesis from long non-coding RNAs following translation of the short open reading frames. Here the authors show that 80S ribosome also guides piRNA production from 3’ UTR of protein-coding genes after translation of long open reading frames, indicating a general piRNA biogenesis mechanism regardless of their precursor ORF length.

8 páginas, 2 figuras, 1 tabla., Ribosome biogenesis is an emerging therapeutic target. It has been proposed that cancer cells are addicted to ribosome production which is therefore considered a druggable pathway in cancer therapy. Cancer cells have been shown to be more sensitive to inhibition of the ribosome production than healthy cells. Initial attempts of inhibiting ribosome biogenesis have been focused on the inhibition of transcription by targeting RNA Pol I. Despite being a promising field of research, several limitations have been identified during the development of RNA Pol I inhibitors, like the lack of specificity or acquired resistance. Ribosome biogenesis is a multistep process and additional points of intervention, downstream the very initial stage, could be investigated. Eukaryotic ribosome maturation involves the participation of more than 200 essential assembly factors that will not be part of the final mature ribosome and frequently require protein-protein interactions to exert their biological action. Using mutagenesis, we have previously shown that alteration of the complex interface between assembly factors impairs proper ribosome maturation in yeast. As a first step toward the developing of ribosome biogenesis inhibitory tools, we have used our previously solved crystal structure of the Chaetomium thermophilum complex between the assembly factors Erb1 and Ytm1 to perform a structure-guided selection of interference peptides. The peptides have been assayed in vitro for their ability to bind their cellular partner using biophysical techniques., Funding was obtained from the Spanish Ministry of Economy, Industry and Competitiveness SAF2017-89901-R and Generalitat Valenciana PROMETEO/2018/055. Open access publication fees have been partially funded by URICI from Consejo Superior de Investigaciones Científicas (CSIC). Molecular graphics and analyses were performed with UCSF Chimera, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from NIH P41-GM103311

Bacterial and eukaryotic hibernation factors prevent translation by physically blocking the decoding center of ribosomes, a phenomenon called ribosome hibernation that often occurs in response to nutrient deprivation. The human pathogen Staphylococcus aureus lacking the sole hibernation factor HPF undergoes massive ribosome degradation via an unknown pathway. Using genetic and biochemical approaches, we find that inactivating the 3′-to-5′ exonuclease RNase R suppresses ribosome degradation in the Δhpf mutant. In vitro cell-free degradation assays confirm that 30S and 70S ribosomes isolated from the Δhpf mutant are extremely susceptible to RNase R, in stark contrast to nucleolytic resistance of the HPF-bound 70S and 100S complexes isolated from the wild type. In the absence of HPF, specific S. aureus 16S rRNA helices are sensitive to nucleolytic cleavage. These RNase hot spots are distinct from that found in the Escherichia coli ribosomes. S. aureus RNase R is associated with ribosomes, but unlike the E. coli counterpart, it is not regulated by general stressors and acetylation. The results not only highlight key differences between the evolutionarily conserved RNase R homologs but also provide direct evidence that HPF preserves ribosome integrity beyond its role in translational avoidance, thereby poising the hibernating ribosomes for rapid resumption of translation. IMPORTANCE Ribosome hibernation is pivotal for the rapid recovery of translation after quiescence in both bacteria and eukaryotes. Ribosome hibernation factors sterically occlude the entry of mRNA and tRNA and are thought to primarily maintain ribosomes in a translation-repressive state, thereby providing a pool of readily recyclable 70S or 80S complexes upon dissociation of the hibernation factors. Ribosomes in Staphylococcus aureus cells lacking the sole hibernation factor HPF are extremely unstable. Here, we show that HPF binding inhibits ribosome degradation by the evolutionarily conserved exoribonuclease RNase R. The data not only uncover a direct protective role of HPF in ribosome stability but also reinforce the versatility of RNase R in RNA processing, decay, and ribosome quality control.

Processing bodies (p-bodies) are a prototypical phase-separated RNA-containing granule. Their abundance is highly dynamic and has been linked to translation. Yet, the molecular mechanisms responsible for coordinate control of the two processes are unclear. Here, we uncover key roles for eEF2 kinase (eEF2K) in the control of ribosome availability and p-body abundance. eEF2K acts on a sole known substrate, eEF2, to inhibit translation. We find that the eEF2K agonist nelfinavir abolishes p-bodies specifically in sensory neurons and impairs translation. To probe the latter, we used cryo-electron microscopy. Nelfinavir stabilizes vacant 80S ribosomes. They contain SERBP1 in place of mRNA and eEF2 in the acceptor site. Phosphorylated eEF2 associates with inactive ribosomes that resist splittingin vitro. Collectively, the data suggest that eEF2 phosphorylation defines a population of inactive ribosomes resistant to recycling and protected from degradation. Thus, eEF2K activity is central to both p-body abundance and ribosome availability in sensory neurons.

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3’UTR correlate with a loss of NMD.Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3’UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3’UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3’UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3’UTR and provide a signal for NMD.